Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5'-deoxyadenosyl radical. In most of these enzymes, the 5'-deoxyadenosyl radical is generated by an iron-sulfur cluster-mediated reductive cleavage of S-adenosyl-(S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt-carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5'-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Radical mechanisms in adenosylmethionine- and adenosylcobalamin-dependent enzymatic reactions

A class of enzymatic reactions of S-adenosylmethionine (AdoMet) has recently been recognized, in which AdoMet plays a novel role by initiating free radical formation through the intermediate formation of 5'-deoxyadenosine-5'-yl, the 5'-deoxyadenosyl radical. The reactions are in this way related to adenosylcobalamin-dependent processes, which also depend on the formation of the 5'-deoxyadenosyl radical as an intermediate. The mechanisms by which the 5'-deoxyadenosyl radical is generated by the AdoMet- and adenosylcobalamin-dependent enzymes are very different. However, the functions of the 5'-deoxyadenosyl radical are similar in that in all cases it abstracts hydrogen from a substrate to form 5'-deoxyadenosine and a substrate-derived free radical. In this paper, the role of the 5'-deoxyadenosyl radical in the reaction of the adenosylcobalamin-dependent reactions will be compared with its role in the AdoMet-dependent reaction of lysine 2,3-aminomutase. The mechanism by which AdoMet is cleaved to the 5'-deoxyadenosyl radical at enzymatic sites will also be discussed.     

S-Adenosyl-L-methionine: beyond the universal methyl group donor

S-Adenosyl-l-methionine (AdoMet or SAM) is a substrate in numerous enzyme-catalyzed reactions. It not only provides methyl groups in many biological methylations, but also acts as the precursor in the biosynthesis of the polyamines spermidine and spermine, of the metal ion chelating compounds nicotianamine and phytosiderophores, and of the gaseous plant hormone ethylene. AdoMet is also the source of catalytic 5'-deoxyadenosyl radicals, produced as reaction intermediates by the superfamily of radical AdoMet enzymes. This review aims to summarize the present knowledge of catalytic roles of AdoMet in plant metabolism.     

Radical SAM activation of the B12-independent glycerol dehydratase results in formation of 5'-deoxy-5'-(methylthio)adenosine and not 5'-deoxyadenosine

Activation of glycyl radical enzymes (GREs) by S-adenosylmethonine (AdoMet or SAM)-dependent enzymes has long been shown to proceed via the reductive cleavage of SAM. The AdoMet-dependent (or radical SAM) enzymes catalyze this reaction by using a [4Fe-4S] cluster to reductively cleave AdoMet to form a transient 5'-deoxyadenosyl radical and methionine. This radical is then transferred to the GRE, and methionine and 5'-deoxyadenosine are also formed. In contrast to this paradigm, we demonstrate that generation of a glycyl radical on the B(12)-independent glycerol dehydratase by the glycerol dehydratase activating enzyme results in formation of 5'-deoxy-5'-(methylthio)adenosine and not 5'-deoxyadenosine. This demonstrates for the first time that radical SAM activases are also capable of an alternative cleavage pathway for SAM.     

Structural diversity in the AdoMet radical enzyme superfamily

AdoMet radical enzymes are involved in processes such as cofactor biosynthesis, anaerobic metabolism, and natural product biosynthesis. These enzymes utilize the reductive cleavage of S-adenosylmethionine (AdoMet) to afford l-methionine and a transient 5'-deoxyadenosyl radical, which subsequently generates a substrate radical species. By harnessing radical reactivity, the AdoMet radical enzyme superfamily is responsible for an incredible diversity of chemical transformations. Structural analysis reveals that family members adopt a full or partial Triose-phosphate Isomerase Mutase (TIM) barrel protein fold, containing core motifs responsible for binding a catalytic [4Fe-4S] cluster and AdoMet. Here we evaluate over twenty structures of AdoMet radical enzymes and classify them into two categories: 'traditional' and 'ThiC-like' (named for the structure of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase (ThiC)). In light of new structural data, we reexamine the 'traditional' structural motifs responsible for binding the [4Fe-4S] cluster and AdoMet, and compare and contrast these motifs with the ThiC case. We also review how structural data combine with biochemical, spectroscopic, and computational data to help us understand key features of this enzyme superfamily, such as the energetics, the triggering, and the molecular mechanisms of AdoMet reductive cleavage. This article is part of a Special Issue entitled: Radical SAM Enzymes and Radical Enzymology.     

Characterization of an allylic analogue of the 5'-deoxyadenosyl radical: an intermediate in the reaction of lysine 2,3-aminomutase.

An allylic analogue of the 5'-deoxyadenosyl radical has been characterized at the active site of lysine 2,3-aminomutase (LAM) by electron paramagnetic resonance (EPR) spectroscopy. The anhydroadenosyl radical, 5'-deoxy-3',4'-anhydroadenosine-5'-yl, is a surrogate of the less stable 5'-deoxyadenosyl radical, which has never been observed but has been postulated to be a radical intermediate in the catalytic cycles of a number of enzymes. An earlier communication [Magnusson, O.Th., Reed, G. H., and Frey, P. A. (1999) J. Am. Chem. Soc. 121, 9764-9765] included the initial spectroscopic identification at 77 K of the radical, which is formed upon replacement of S-adenosylmethionine by S-3',4'-anhydroadenosylmethionine as a coenzyme for LAM. The electron paramagnetic resonance spectrum of the radical changes dramatically between 77 and 4.5 K. This unusual temperature dependence is attributed to a spin-spin interaction between the radical and thermally populated, higher spin states of the [4Fe-4S]+2 center, which is diamagnetic at 4.5 K. The EPR spectra of the radical at 4.5 K have been analyzed using isotopic substitutions and simulations. Analysis of the nuclear hyperfine splitting shows that the unpaired spin is distributed equally between C5'- and C3'- as expected for an allylic radical. Hyperfine splitting from the beta-proton at C-2'(H) shows that the dihedral angle to the p(z)-orbital at C-3' is approximately 37 degrees. This conformation is in good agreement with a structural model of the radical. The rate of formation of the allylic radical shows that it is kinetically competent as an intermediate. Measurements of 2H kinetic isotope effects indicate that with lysine as the substrate, the rate-limiting steps follow initial reductive cleavage of the coenzyme analogue.     

Radical SAM enzymes involved in the biosynthesis of purine-based natural products

The radical S-adenosyl-l-methionine (SAM) superfamily is a widely distributed group of iron-sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5'-deoxyadenosyl radical, which is produced by the reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F(420). This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

S-adenosylmethionine radical enzymes

The role of S-adenosylmethionine (SAM) as a precursor to organic radicals, generated by one-electron reduction of SAM and subsequent fission to form 5'-deoxyadenosyl radical and methionine, has been known for some time. Only recently, however, has it become apparent how widespread such enzymes are, and what a wide range of chemical reactions they catalyze. In the last few years several new SAM radical enzymes have been identified. Spectroscopic and kinetic investigations have begun to uncover the mechanism by which an iron sulfur cluster unique to these enzymes reduces SAM to generate adenosyl radical. Most recently, the first X-ray structures of SAM radical enzymes, coproporphyrinogen-III oxidase, and biotin synthase have been solved, providing a structural framework within which to interpret mechanistic studies.     

S-adenosylmethionine as an oxidant: the radical SAM superfamily

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.     

S-adenosylmethionine-dependent enzyme activation

S-Adenosylmethionine (SAM)-dependent activations of pyruvate formate-lyase, lysine 2,3-aminomutase and cobalamin-dependent methionine synthase are discussed. In each case, cleavage of SAM is accompanied by the formation of a catalytically active enzyme. The chemistry of activation of these three enzymes falls into three distinct classes: generation of an essential enzyme radical (pyruvate formate-lyase), formation of a catalytically active 5'-deoxyadenosyl radical (lysine 2,3-aminomutase) and reductive methylation to form a required methylcobalamin complex (methionine synthase).     

Self-sacrifice in radical S-adenosylmethionine proteins

The radical SAM superfamily of metalloproteins catalyze the reductive cleavage of S-adenosyl-l-methionine to generate a 5'-deoxyadenosyl radical (5'-dA*) intermediate that is obligate for turnover. The 5'-dA* acts as a potent oxidant, initiating turnover by abstracting a hydrogen atom from an appropriate substrate. A special class of these enzymes use this strategy to functionalize unactivated C-H bonds by insertion of sulfur atoms. This review will describe the characterization of three members of this class - biotin synthase, lipoyl synthase, and MiaB protein - each of which has been shown to cannibalize itself during turnover.     

Radicals from S-adenosylmethionine and their application to biosynthesis

The radical SAM superfamily of enzymes catalyzes a broad spectrum of biotransformations by employing a common obligate intermediate, the 5'-deoxyadenosyl radical (DOA). Radical formation occurs via the reductive cleavage of S-adenosylmethionine (SAM or AdoMet). The resultant highly reactive primary radical is a potent oxidant that enables the functionalization of relatively inert substrates, including unactivated C-H bonds. The reactions initiated by the DOA are breathtaking in their efficiency, elegance and in many cases, the complexity of the biotransformation achieved. This review describes the common features shared by enzymes that generate the DOA and the intriguing variations or modifications that have recently been reported. The review also highlights selected examples of the diverse biotransformations that ensue.     

Radical AdoMet enzymes in complex metal cluster biosynthesis

Radical S-adenosylmethionine (AdoMet) enzymes comprise a large superfamily of proteins that engage in a diverse series of biochemical transformations through generation of the highly reactive 5'-deoxyadenosyl radical intermediate. Recent advances into the biosynthesis of unique iron-sulfur (FeS)-containing cofactors such as the H-cluster in [FeFe]-hydrogenase, the FeMo-co in nitrogenase, as well as the iron-guanylylpyridinol (FeGP) cofactor in [Fe]-hydrogenase have implicated new roles for radical AdoMet enzymes in the biosynthesis of complex inorganic cofactors. Radical AdoMet enzymes in conjunction with scaffold proteins engage in modifying ubiquitous FeS precursors into unique clusters, through novel amino acid decomposition and sulfur insertion reactions. The ability of radical AdoMet enzymes to modify common metal centers to unusual metal cofactors may provide important clues into the stepwise evolution of these and other complex bioinorganic catalysts. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores' extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the CS bond associated with the sulfonium ion in SAM, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in Bacillus subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5'-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.     

Adenosylmethionine as a source of 5'-deoxyadenosyl radicals

The combination of an iron-sulfur cluster and S-adenosylmethionine provides a novel mechanism for the initiation of radical catalysis in an unanticipated variety of metabolic processes. Molecular details of the cluster-mediated reductive cleavage of S-adenosylmethionine to methionine and, presumably, a 5'-deoxyadenosyl radical are the targets of recent studies.     

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes

'Radical SAM' enzymes generate catalytic radicals by combining a 4Fe-4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 A resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A) and a neighboring sulfur of the cluster (3.6 A), allowing single electron transfer from the 4Fe-4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5'-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.     

Complex biotransformations catalyzed by radical S-adenosylmethionine enzymes

The radical S-adenosylmethionine (AdoMet) superfamily currently comprises thousands of proteins that participate in numerous biochemical processes across all kingdoms of life. These proteins share a common mechanism to generate a powerful 5'-deoxyadenosyl radical, which initiates a highly diverse array of biotransformations. Recent studies are beginning to reveal the role of radical AdoMet proteins in the catalysis of highly complex and chemically unusual transformations, e.g. the ThiC-catalyzed complex rearrangement reaction. The unique features and intriguing chemistries of these proteins thus demonstrate the remarkable versatility and sophistication of radical enzymology.     

Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced [4Fe-4S](+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.